WO2017219916A1

WO2017219916A1 - Molecule, cell expressing the same, and preparation method and use thereof

Info

Publication number: WO2017219916A1
Application number: PCT/CN2017/088594
Authority: WO
Inventors: 李鹏; 魏新茹; 叶未; 赵若聪; 赖允鑫; 林思妙; 姚瑶
Original assignee: 深圳市体内生物医药科技有限公司
Priority date: 2016-06-20
Filing date: 2017-06-16
Publication date: 2017-12-28
Also published as: CN113512126B; CN113512126A; CN107624119A; CN107522786A; CN107624119B

Abstract

Provided are a chimeric immune inhibitory checkpoint receptor molecule, a nucleic acid encoding the same, a construct, expression vector and transformed cell containing the nucleic acid, and a pharmaceutical use thereof. The chimeric immune inhibitory checkpoint receptor molecule comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain and the optional transmembrane domain are corresponding domains of an immune inhibitory checkpoint receptor molecule or are genetically engineered on the basis of the corresponding domains. The intracellular domain is one or more immune activation signal domains. The transformed cell has a strong tumor-killing effect and can abrogate a tumor-immunosuppressive microenvironment, thereby strengthening an immune response in the tumor microenvironment. The transformed cell also has a self-regulation function and can alleviate side effects such as an overactive, non-specific immune response and a cytokine storm.

Description

Molecule, cell expressing the same, preparation method and use thereof

Technical field

The present invention relates to the field of cellular immunotherapy technology for tumors, and in particular to a chimeric immunosuppressive checkpoint receptor molecule, a nucleic acid encoding the same, a construct containing the nucleic acid, an expression vector and transformed cells, and Pharmaceutical use.

Background technique

Current status of CAR-T cell tumor therapy

In October 2015, the National Cancer Center released the cancer data of residents in China for the first time in the famous cancer professional journal "Cancer Letters". The results showed that the number of cases diagnosed as cancer in China within 5 years and still survivable was about 7.49 million (including 3.86 million male patients and 3.81 million female patients). The overall 5-year cancer prevalence rate was 556/100,000. It reflects the urgent need for cancer patients to progress in new treatment research.

Immunotherapy has become a new hope for humans to fight cancer, and the clinical trial results of CAR-T in the treatment of hematological tumors, especially B-ALL leukemia, have made it one of the most well-received tumor immunotherapy in recent years. However, the CAR T immunotherapy research targeting solid tumors still faces a huge test: how to break through the solid tumor tissue barrier, how to resist the immunosuppression in the tumor tissue, and how the immune cells compete for survival in the hypoxic microenvironment.

The problem of tumor treatment, especially the immunosuppression in solid tumors and their tumor microenvironment

The tumor microenvironment can down-regulate the number of tumor antigen-specific helper/toxic killer cells and the release of inflammatory factors, and up-regulate the number and inhibitory functions of immunosuppressive cells, such as regulatory T cells and myeloid-derived suppressor cells ( Myeloid-Derived Suppressor Cells) subvert the normal immune system as a dominant factor in promoting tumor growth.

Status of corresponding measures against tumor immunosuppression

In response to the immunosuppression of solid tumors, scientists have successively developed a blocking inhibition signaling pathway to down-regulate tumor microscopy. A method for inhibiting cells in the environment, such as specifically targeting PD-1 (Programmed Death 1), PD-L1 (Programmed Death Ligand 1), CTLA4 (Cytotoxic T-Lymphocyte-associated Antigen-4) antibody or inhibitor, Immune activators and the like.

In the prior art, the application of the immunosuppressive-associated antigens PD-1 and CTLA-4 to tumor immunotherapy is usually to construct a PD-1 antibody (Pembrolizumab, Nivolumab) or a CTLA-4 antibody (Ipilimumab), or use PD- 1 or an inhibitor of CTLA-4 or a signaling pathway blocker. However, PD-1 antibodies and CTLA-4 antibodies have certain limitations, that is, antibodies need to penetrate into tumor tissues to exert therapeutic effects, so the clinical application of antibodies is uncertain. It has been reported that when CAR-GD2T cells are combined with PD-1 antibody for immunotherapy, they can resist the immunosuppressive effect of the tumor microenvironment and enhance the tumor killing of CAR T cells. For immunotherapeutic applications of PD-1 and CTLA-4, co-expression of targeting tumor-associated antigens CAR T and inhibitory CAR T has also been reported in the prior art (the extracellular antigen is a normal cell antigen and the intracellular domain is PD-1). Or the inhibitory signal domain of CTLA-4) prevents the occurrence of CAR T off-target (off-target phenomenon: CAR T cells recognize and kill normal cells).

In addition, CAR T co-expressing the diabody PD-1 and CD19/Mesothelin/PSCA has also been reported in the prior art, although this expands the target range (expressing PD-1 or CD19/Mesothelin/PSCA or both) Tumor cells), but neglecting the side effects caused by blindly increasing cell killing: cytokine storm, off-target phenomenon (normal cells also express two antigens or one, causing CAR T to kill normal cells, causing functional defects in normal cell loss) ).

Summary of the invention

It is an object of the present invention to provide a chimeric immunosuppressive checkpoint receptor molecule, a nucleic acid encoding the same, a construct comprising the nucleic acid, an expression vector and transformed cells, and their pharmaceutical use. The transformed cell expressing the chimeric immunosuppressive checkpoint receptor molecule of the present invention can create a tumor immunosuppressive microenvironment with enhanced tumor killing effect; and the transformed cell has self-regulating function and can be alleviated Excessive side effects such as non-specific immune effects and cytokine storms.

The present invention achieves the above objects by the following technical solutions:

In a first aspect, the invention provides a chimeric immunosuppressive checkpoint receptor molecule comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain and optionally The transmembrane domain is the corresponding domain of an immunosuppressive checkpoint receptor molecule or is genetically engineered based on the domain, the intracellular domain being one or more immune activation signal domains.

Preferably, the immunosuppressive checkpoint receptor molecule is selected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, A2AR, B7H3, B7H4, BTLA, IDO, KIR, CD160, 2B4 (CD244) and VISTA (C10orf54);

Further preferably, the immune checkpoint receptor molecule is PD-1, CTLA-4, LAG3 or TIM3.

Preferably, the intracellular domain comprises an immunostimulatory signal combination and an optional signal peptide;

Further preferably, said immune costimulatory signal combination comprises a TLR1 and/or TLR2 signal domain;

Further preferably, the intracellular domain is a combination of TLR1 and/or TLR2 and a 41BB and/or CD28 signaling domain.

Further preferably, the optional signal peptide is a signal peptide corresponding to an immunosuppressive checkpoint receptor molecule.

Further preferably, the intracellular domain further comprises a CD3 sputum intracellular domain;

In a specific preferred embodiment, the TLR1 and/or TLR2 signal domain and optionally the 41BB and/or CD28 signal domain are arranged on the N-terminal side of the CD3 cell intracellular domain.

In a second aspect, the invention provides a nucleic acid encoding a chimeric immunosuppressive checkpoint receptor molecule of the first aspect.

In a third aspect, the present invention provides a nucleic acid construct comprising the nucleic acid of the second aspect, and an immunosuppressive checkpoint receptor molecule operably linked thereto for directing said chimerism in a host cell One or more control sequences expressed.

In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid construct of the third aspect; preferably, the expression vector is a lentiviral expression vector.

In a fifth aspect, the present invention provides a transformed cell comprising the nucleic acid of the second aspect, or wherein the nucleic acid construct of the third aspect or the expression vector of the fourth aspect is transformed;

Preferably, the transformed cell is an immune cell, further preferably a T cell, a B cell or an NK cell.

In a specific embodiment, the transformed cell is a Zeus sulphate cytotoxic T lymphocyte (S-CTL), which is a lymphocyte that overexpresses a checkpoint molecule that undergoes immunosuppression to activation, particularly T lymphocytes. S-CTL cells include SP-CTL cells expressing a chimeric PD-1 receptor molecule, SC-CTL cells expressing a chimeric CTLA-4 receptor molecule, and ST-CTL cells expressing a chimeric TIM3 receptor molecule. And SL-CTL cells expressing a chimeric LAG3 receptor molecule and the like. S-CTL cells obtain lymphocytes resistant to immunosuppression by transfecting a chimeric immunosuppressive-agonistic transition checkpoint molecule gene into human T cells. S-CTL lymphocytes have the following characteristics:

1) potent cell killing effect on tumor cells or tumor-associated immunosuppressive cells expressing ligands of immunosuppressive checkpoint molecules (such as PD-L1, B7.1, B7.2, Galectin-9, MHC II, etc.);

2) S-CTL contains natural immunosuppressive checkpoint receptor molecules (such as PD-1, CTLA-4, TIM3, LAG3, etc.) expressed on the surface of immune effector cells, especially T cells, NK cells, etc., and tumor cells or tumors. After binding of the ligand of the relevant immunosuppressive cell, the immune activation signal downstream of the S-CTL vector can be activated to promote the activation, proliferation, recognition, killing and other functions of the immune cell itself;

3) S-CTL structure: extracellular segment and transmembrane region are completely natural immunosuppressive checkpoint molecular structures for specific recognition of tumor cell surface ligands and recognition signals for efficient delivery in S-CTL cells; intracellular The region is one or more immune activation signal domains, wherein the present invention finds an intracellular domain comprising a TLR1 and/or TLR2 signaling activation domain, which further promotes tumor killing effects;

4) Self-regulating function, since normal lymphocytes express natural immunosuppressive checkpoint molecular receptors, transformed cells express chimeric immunosuppressive-agonistic transition checkpoint molecules, while natural immunosuppressive checkpoint molecular receptors are conductive Inhibition of the signal, the chimeric immunosuppressive checkpoint molecule receptor is a conduction activation signal, thereby allowing immunity The effector cells themselves perform feedback expression regulation and slow down the side effects caused by excessive activation of S-CTL cells, such as off-target killing and cytokine storm;

In a sixth aspect, the invention provides a method of producing a transformed cell of the fifth aspect, comprising: introducing a nucleic acid of the second aspect into a cell, or transforming the nucleic acid construct of the third aspect Or the step of expressing the vector as described in the fourth aspect.

In a seventh aspect, the invention provides a chimeric immunosuppressive checkpoint receptor molecule according to the first aspect, a nucleic acid according to the second aspect, a nucleic acid construct according to the third aspect, the fourth aspect The expression vector or the transformed cell of the fifth aspect, in the preparation of a ligand corresponding to an immunosuppressive checkpoint receptor molecule, preferably with PD-L1, B7.1, B7.2, Galectin-9, MHC Use of a drug for expression of a disease associated with II;

Preferably, the disease is a tumor corresponding to a ligand expressing an immunosuppressive checkpoint receptor molecule, preferably a tumor expressing PD-L1, B7.1, B7.2, Galectin-9, MHC II;

Further preferably, the tumor corresponding to the immunosuppressive checkpoint receptor molecule, preferably expressing PD-L1, B7.1, B7.2, Galectin-9, MHC II, is selected from the group consisting of lung cancer, leukemia, breast cancer, Prostate cancer, pancreatic cancer, liver cancer, melanoma, and non-melanoma skin cancer.

Definition of Terms

In the context of the present invention, an "Immune Checkpoint" is a molecule that up-regulates or down-regulates an immunostimulatory signal in the immune system, and is classified into a Stimulatory Checkpoint and an Inhibitory Checkpoint molecule. ).

An "immunosuppressive checkpoint" is a molecule that down-regulates an immune stimulus signal in the immune system, such as: PD-1, CTLA-4, TIM3, LAG3, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, CD160, 2B4 (CD244), VISTA (C10orf54), TIGIT, LAIR1, etc.

"Immune activation signal": During lymphocyte activation, T cells need to acquire two signals for full activation; the first signal is antigen-specific, that is, through the T cell receptor and the antigen-presenting cell APC surface MHC molecule junction The second signal, the costimulatory signal, is antigen-unspecific and is produced by the binding of T cells and costimulatory signaling molecules on the surface of APC cells.

The intracellular domain of the chimeric immunosuppressive checkpoint receptor molecule of the invention may comprise costimulatory signaling molecules CD28, OX40, ICOS (CD278), 4-1BB (CD137), CD40, CD27, CD134, CDS, ICAM- 1. Intracellular segments of LFA-1 (CD11a/CD18), CD2, BTLA, etc., or any combination thereof.

Beneficial effect

The transformed cell of the present invention is a novel tumor killer cell which specifically recognizes cells expressing an immunosuppressive checkpoint molecular ligand in a tumor suppressing immunosuppressive microenvironment, has a low off-target rate; and, its intracellular domain The unique design can further promote its tumor killing effect, and can effectively eliminate the tumor immunosuppressive effect; Moreover, the immune effector cells (such as T cells, etc.) transformed by the present invention are induced to express a certain amount of natural immunosuppression in the tumor microenvironment. Checkpoint receptor molecules, which bind to tumor cells, tumor-associated immunosuppressive cells, or immune-expressed antigens to produce a certain feedback inhibition regulatory signal, which has a certain regulatory effect on the immune effects produced by transformed cells, ie, does not cause The transformed cells are transiently activated, causing discomfort such as inflammation. In addition, the activated vector transformed by the present invention is optimized for immune activation, and comprises an intrinsic immune activation signal TLR1/TLR2 intracellular segment, and a combination of 4-1BB, CD28 and other immune activation molecules to construct a co-stimulatory signal, and finally a CD3 ζ chain The construction of complete immune signaling has a strong immune activation effect. S-CTL cells have strong functions of killing tumor cells and tumor-associated immunosuppressive cells. For example, SC-CTL cells can specifically recognize the microenvironment in killing tumors. CD80+/CD86+ tumor cells and tumor-associated immunosuppressive cells synergistically promote anti-tumor immune effects.

In view of the above advantages, the transformed cells expressing the chimeric receptor molecule of the present invention have a good clinical application prospect in antitumor therapy.

DRAWINGS

Figure 1 shows the synthetic sequence map in Example 1.

Figure 2 shows the pWPXLd-S(PD1)-CTL-2A-EGFP plasmid constructed in Example 1, pWPXLd-S(CTLA-4)-CTL-2A-EGFP plasmid, pWPXLd-S(TIM3)-CTL-2A- EGFP plasmid and pWPXLd-S (LAG3)-CTL-2A-EGFP plasmid map.

Figure 3 shows the positive rate of viral infection of SP-CTL cells by flow cytometry; blank group: non-transfected T cells stained without CD3 flow cytometry.

Figure 4 shows the positive rate of viral infection of SC-CTL cells by flow cytometry; normal T cell group: non-transfected T cells.

Figure 5 shows the killing effect of SP-CTL cells on the human T-ALL cell line JURKAT-GL.

Figure 6 shows the killing effect of SP-CTL cells on human non-small cell lung cancer H460-GL.

Figure 7 shows the killing effect of SC-CTL cells on the human B-ALL cell line NALM6-GL.

Figure 8 shows the in vivo killing effect of SP-CTL cells on human non-small cell lung cancer H460-GL.

Figure 9 shows the in vitro killing effect of SP-CTL cells on A549 cells that do not express PD-L1.

Figure 10 shows the in vitro killing effect of SP-CTL cells on K562 cells that do not express PD-L1.

Figure 11 is a graph showing the results of flow cytometry detection of PD-L1 expression in lung cancer cell lines H1299, H460, H23 and A549 and prostate cancer cell line PC3.

Figure 12 shows the in vitro killing effect of SP-CTL cells on PD-L1 expressing lung cancer cell lines H1299, H460, H23 and A549 and prostate cancer cell line PC3.

Figure 13 is a graph showing the results of in vivo recognition and killing of primary lung cancer cells by SP-CTL cells; wherein Fig. 13-A shows the average volume results of tumor tissue blocks in each experimental group at different time points, and Fig. 13-B shows each experimental group. The size of the three replicated tumor tissue blocks is aligned with the picture.

Figure 14 is a graph showing the results of in vivo recognition and killing of primary gastric cancer cells by SP-CTL cells; wherein Figure 14-A shows the size comparison of three replicate tumor tissue blocks in each experimental group, and Figure 14-B shows the experimental groups. The average weight result of the tumor tissue mass.

Figure 15 is a graph showing the results of in vivo recognition and killing of primary osteosarcoma cells by SP-CTL cells; wherein Figure 15-A shows a size comparison of six replicate tumor tissue blocks in each experimental group, and Figure 15-B shows each experiment. The average weight result of the group of tumor tissue blocks.

Figure 16 is a graph showing the results of in vitro specific recognition and killing of non-Hawsonson lymphoma RL cells by SC-CTL cells.

Figure 17 is a graph showing the results of in vivo recognition and killing of non-Hawking's lymphoma RL cells by SC-CTL cells; Figure 17-A shows a comparison of the size of two replicate tumor tissue blocks in each experimental group, Figure 17- B shows the average weight result of the tumor tissue mass of each experimental group.

Figure 18 is a graph showing the results of in vivo recognition and killing of primary osteosarcoma cells by mSC-CTL cells; wherein Figure 18-A shows the size comparison of the tumor tissue blocks of each experimental group, and Figure 18-B shows the experimental groups. The average weight result of the tumor tissue mass.

Figure 19 is a graph showing the results of SP-CTL, mSC-CTL alone or in combination to specifically recognize and kill primary lung cancer cells in vivo; Figure 19-A shows the average volume results of tumor tissue blocks in each experimental group, Figure 19-B The average weight results of the tumor tissue blocks of each experimental group are shown.

Figure 20 shows that SP-CTL, mSC-CTL alone or in combination with CD80+ cells (Fig. 20-A), CD86+ cells (Fig. 20-B) and CD11b+Ly6G+ cells (MDSC) in tumor tissues in vivo (Fig. 20- C) The impact.

Figure 21 shows that the B acute lymphoblastic leukemia cell line Nalm6, RL, Raji highly expresses the Lag3 ligand MHC II.

Figure 22 shows that SL-CTL cells specifically recognize and kill the B-ALL cell line Nalm6 cells that highly express MHC II ligand.

Figure 23 shows that SL-CTL cells co-incubated with tumor cells significantly up-regulated protein expression and secretion of inflammatory factors IL-2 (Figure 23A), IFN-γ (Figure 23A).

Figure 24 shows that A549 cells do not express Galectin9 (Tim3 ligand) surface protein in vitro, but in vivo micro The environment-induced lung cancer cell line A549 specifically expresses Galectin9 surface protein.

Figure 25 shows that primary

lung cancer cells

1, 2, and 3 specifically express Galectin9 surface protein in a patient-derived tumor xenograft (PDX) model.

Figure 26 shows that ST-CTL specifically recognizes primary lung cancer cells that kill high expression of Galectin9 surface protein in the PDX model.

detailed description

To facilitate an understanding of the invention, the invention is set forth below. It should be understood by those skilled in the art that the present invention is not to be construed as limited.

Example 1. Preparation of S-CTL cells

Plasmid construction

1) Through gene synthesis, "immunosuppressive checkpoint molecule (signal peptide + extracellular segment + transmembrane region) - immunostimulatory signal 1 intracellular segment - immunostimulatory signal 2 (generally TLR1 or TLR2) intracellular segment - CD3 得到The DNA sequence, the first and the end of the DNA sequence contain the PmeI and Spe1 restriction sites, respectively. The synthetic sequence map is shown in Figure 1.

For the gene sequence synthesized in the examples of the present invention, see SEQ NO. 1 (for the preparation of S-CTL targeting PD-1, ie, SP-CTL, the synthesized DNA sequence, the structure is PD-1 signal peptide + extracellular Segment + transmembrane region - CD28-TLR1/2-CD3ζ) and SEQ NO. 2 (for the preparation of S-CTL targeting CTLA-4, ie, SC-CTL, the synthesized DNA sequence, the structure is CTLA-4 signal peptide + extracellular segment + transmembrane region - CD28 intracellular segment - TLR1/2 intracellular segment - CD3 ζ) and SEQ NO. 3 (for the preparation of S-CTL targeting TIM3, ie, ST-CTL, the synthesized DNA sequence, The structure is TIM3 signal peptide + extracellular domain + transmembrane region - 4-1BB intracellular segment - TLR2 intracellular segment - CD3 ζ) and SEQ NO. 4 (for the preparation of S-CTL targeting LAG3, ie SL-CTL, The synthesized DNA sequence has the structure of LAG3 signal peptide + extracellular segment + transmembrane region - 4-1BB intracellular segment - TLR2 intracellular segment - CD3 ζ).

S-CTL (Zeushield Cytotoxic T Lymphocyte) cells, which are genetically modified to overexpress S-CTL chimera Receptors are T lymphocytes that enhance immune killing ability, and are not limited to T cells, and immune effector cells overexpressing S-CTL chimeric receptors also have enhanced immunological killing function. S-CLT chimeric receptors, ie chimeric immunosuppressive checkpoint molecules, extracellular and transmembrane segments are natural human immunosuppressive checkpoint receptor molecules such as PD-1, CTLA-4, TIM3, LAG3, A2AR , B7-H3, B7-H4, BTLA, IDO, KIR, CD160, 2B4 (CD244), VISTA (C10orf54), TIGIT, LAIR1, etc., and the intracellular segment is an immunostimulatory signaling molecule comprising TLR1/TLR2 Combination with intracellular segments such as TLR3-10, 4-1BB, CD28, ICOS, CD27, CD2, OX40, CD30, CD40, LFA-1, CD7, CD2S, LIGHT, NKG2C, B7-H3, CD83, etc. The CD3zeta chain is composed of a combination; it can specifically recognize and kill tumor cells and tumor-associated immunosuppressive cells expressing immunosuppressive checkpoint ligands.

SP-CTL, an S-CTL cell expressing a chimeric PD-1 receptor, specifically recognizes tumor cells and tumor-associated stromal cells or immunosuppressive cells that secrete PD-L1.

SC-CTL, a S-CTL cell expressing a chimeric CTLA-4 receptor, specifically recognizes tumor cells and tumor-associated stromal cells or immunosuppressive cells that kill B7.1 and/or B7.2.

ST-CTL, an S-CTL cell expressing a chimeric TIM3 receptor, specifically recognizes tumor cells and tumor-associated stromal cells or immunosuppressive cells that secrete Galectin-9.

SL-CTL, a S-CTL cell expressing a chimeric LAG3 receptor, specifically recognizes tumor cells that secrete MHC II and tumor-associated stromal cells or immunosuppressive cells.

2) Using the Thermo restriction enzymes Pme1 and Spe1, the synthetic S-CTL DNA fragments (SEQ NO. 1, SEQ NO. 2, SEQ NO. 3, SEQ NO. 4) and pWPXLd- were respectively obtained by double digestion. Linearized DNA (containing sticky ends) of the EGFP vector.

3) Recovery by agarose gel electrophoresis, linearized synthetic S-CTL (SEQ NO. 1, SEQ NO. 2, SEQ NO. 3, SEQ NO. 4) and pWPXLd-EGFP vector DNA fragment with sticky ends were obtained.

4) Linked linearized SEQ NO.1 by T4 DNA ligase (from Invitrogent) pWPXLd-S(PD1/CTLA-4/TIM3/LAG3)-CTL-2A-EGFP plasmid was obtained with pWPXLd-2A-EGFP, linearized SEQ NO.2 and pWPXLd-2A-EGFP, and the plasmid map is shown in the attached drawing. 2.

S-CTL Lentiviral Packaging

1) Incubate 293T cells to a density of 80-90%, and replace the medium with: DMEM high glucose medium + 1% FBS + 1% double antibody;

2) After 2-6 hours, the pWPXLd-S (PD1/CTLA-4/TIM3/LAG3)-CTL-2A-EGFP plasmid was transfected into the 293T together with the lentiviral helper plasmid (pMD2.G, psPAX2) with PEI. The cells were co-transfected with the empty vector pWPXLd-EGFP and the helper plasmid as a control group;

3) The culture supernatant, S(PD1/CTLA-4/TIM3/LAG3)-CTL virus supernatant, was collected at 24, 48 and 72 hours after transfection, and stored at -80 °C until use.

T cell activation and lentiviral infection

1) separating monocytes in blood by Ficoll density gradient method or red blood cell lysis method;

2) T cells were sorted by MACS Pan-T magnetic beads and diluted to a concentration of 2.5×10 ⁶ with T cell medium (AIM-V plus 5% FBS, penicillin 100 U/ml and streptomycin 0.1 mg/ml). /ml to be used;

3) stimulating T cells by magnetic beads coated with CD2, CD3, and CD28 antibodies, and stimulating for 48 hours in an incubator at 37 ° C under 5% CO ₂ ;

4) remove the magnetic beads in the T cells, centrifuge to remove the supernatant, and resuspend;

5) Add S (PD1/CTLA-4/TIM3/LAG3)-CTL lentiviral supernatant and 8 μg/ml of polybrene and 300 IU/ml IL-2, the amount of virus added is MOI=10, at 37 ° C, 5% CO ₂ Culture in an incubator under conditions;

6) After 24 h, centrifuge at 300 g for 5 min, remove the supernatant, resuspend in fresh T cell medium containing 300 IU/ml IL-2, and incubate in an incubator at 37 ° C under 5% CO ₂ for 2-3 Half a day of liquid change;

7) The ratio of S-CTL (SC-CTL or ST-CTL) of GFP-positive T cells was detected by flow cytometry, and the results are shown in Fig. 3 and Fig. 4.

Example 2: In vitro killing effect of SP-CTL cells on human leukemia cell line JURKAT

1) Transduction of luciferase (Luciferace) in the leukemia cell line JURKAT to construct a JURKAT-GL reporter cell line;

2) SC-CTL GFP-positive cells and JURKAT-GL cells were mixed and cultured in a ratio of 1:2 (the control group was GFP T cells, the blank group was no T cells, and each group was set with three duplicate wells). ;

3) After 18 hours, gently pipette a half volume of the medium supernatant, add an equal volume of luciferase substrate (concentration: 300 μg / ml), and mix;

4) The RLU (relative light unit) was measured by a multi-function microplate reader, and the measurement time was set to 1 second.

5) Formula for killing ratio: 100% × (blank hole reading - experimental hole reading) / blank hole reading, the in vitro killing ratio of SP-CTL cells to human leukemia cell line JURKAT is shown in FIG. 5 .

Example 3: In vitro killing effect of SP-CTL cells on non-small cell lung cancer H460

1) Transduction of luciferase (Luciferace) in non-small cell lung cancer H460 to construct a H460-GL reporter cell line;

2) SP-CTL GFP-positive cells and H460-GL cells were mixed and co-cultured at a ratio of 1:2 (the control group was GFP T cells, the blank group was no T cells, and each group was set with three duplicate wells). ;

5) Formula for killing ratio: 100% × (blank hole reading - experimental hole reading) / blank hole reading (Figure 4), the ratio of SP-CTL cells to non-small cell lung cancer H460 in vitro is shown in Figure 6. .

Example 4: In vitro killing effect of SC-CTL cells on human leukemia cell line NALM6

1) Transduction of luciferase (Luciferace) in the leukemia cell line NALM6 to construct a NALM6-GL reporter cell line;

2) SC-CTL virus-infected mixed cells with 1×10 of GFP-positive cells were 3.5×10 ⁴ , 1.75×10 ⁴ , 8.6×10 ³ , 4.4×10 ³ , 2.2×10 ³ , and 1.1×10 ^{3 , respectively} . ^Four NALM6-GL cells were co-cultured (the control group was GFP T cells, the blank group was no T cells, and each group was set with three duplicate wells);

5) Killing ratio calculation formula: 100% × (blank hole reading - experimental hole reading) / blank hole reading, the in vitro killing ratio of SC-CTL cells to human leukemia cell line NALM6 is shown in FIG. 7 .

Example 5: In vivo killing effect of SP-CTL cells on human non-small cell lung cancer H460

1) 1×10 ⁵ H460-GL cells were subcutaneously transplanted into immunodeficient mice NOD/SCID IL2rg-/-;

2) 10 days after injection of the H460-GL tumor bearing mice SP-CTL GFP positive cells to SP-CTL 4 × 10 ⁶ virus infected cells were mixed, 15 days after the second injection of 4 × 10 ⁶ SP-CTL The virus infects the mixed cells, and the control group is the tumor-bearing mice injected with GFP T cells in the same operation as above, and three replicates are set for each experimental group;

3) After tumor transplantation, the volume of H460 tumor mass was measured on

days

10, 14, 17, 20, 23, 26, 29, 32, and 35, and the results are shown in Fig. 8.

From the above test results, it is understood that the SP-CTL cells of the present invention have significant in vivo and exogenous killing effects on various tumors expressing the corresponding ligands.

Example 6, safety experiment of SP-CTL cells

1) Transduction of luciferase (Luciferace) in lung adenocarcinoma cell line A549 and chronic myeloid leukemia cell line K562 which do not express ligand PD-L1, and construction of A549-GL, K562-GL reporter cell line;

2) SP-CTL GFP-positive cells were co-cultured with A549-GL and K562-GL cells at a ratio of 1:2 (the control group was GFP T cells, and three replicate wells were set in each group);

3) After 18 hours, gently pipette half of the medium supernatant and add an equal volume of luciferase Substrate (concentration: 300 μg/ml), mix;

4) The in vitro killing ratio of SP-CTL cells to A549-GL and K562-GL cells was detected by a multi-function microplate reader, and the results are shown in Figures 9 and 10, respectively.

It can be seen from the above results that SP-CTL cells have a specific killing effect only on target cells expressing PD-L1, no killing effect on cells not expressing PD-L1, and have stable biosafety.

Example 7, SP-CTL cells were identified in vitro to kill lung cancer and prostate cancer cells

1) The expression of ligand PD-L1 in lung cancer cell lines (H1299, H460, H23, A549) and prostate cancer cell line (PC3) cells was detected by flow cytometry, and the results are shown in Fig. 11;

2) SP-CTL GFP-positive cells were separately labeled with H1299, H460, H23, A549, and PC3 cells labeled with luciferase GL (H1299-GL, H460-GL, H23-GL, A549-GL, PC3-GL, respectively) The cells were co-cultured in a ratio of 1:1, 1:2, 1:4, 1:8, 1:16 (the control group was GFP T cells, and three replicate wells were set in each group);

4) The in vitro killing ratio of SP-CTL cells to H1299-GL, H460-GL, H23-GL, A549-GL and PC3-GL cells was detected by a multi-function microplate reader. The results are shown in Fig. 12.

From the above test results, SP-CTL cells can specifically recognize and kill target cells (lung cancer cells, prostate cancer cells, etc.) expressing PD-L1.

Example 8, SP-CTL cells recognize killer primary lung cancer cells in vivo

1) Cut the primary lung cancer tissue sample into small pieces of tumor tissue with a diameter of 3 mm, and transplant it into the immunodeficient mouse NOD/SCID IL2rg-/- to construct a lung cancer mouse model.

2) 20 days after injection of lung cancer in a mouse model of SP-CTL of GFP positive cells of 4 × 10 ⁶ SP-CTL infected cells were mixed, 25 days of the second injection of 4 × 10 ⁶ SP-CTL mixed infection The cells and the control group were tumor-bearing mice injected with GFP T cells, and three replicates were set for each experimental group;

3) On the 20th, 24th, 28th, 20th, 23rd, 26th, 29th, 32th and 35th day after tumor transplantation, the volume of subcutaneous tumor tissue in lung cancer was measured; the average volume of tumor tissue blocks in each experimental group at different time points The result is shown in Figure 13-A.

4) Tumor tissue blocks were obtained from the mouse model of lung cancer on the 44th day after tumor transplantation; the size alignment of the three replicated tumor tissue blocks in each experimental group is shown in Fig. 13-B.

From the above test results, it can be seen that the SP-CTL cells of the present invention can effectively eliminate primary lung cancer cells in vivo in the case of primary tumor heterogeneity and immunosuppressive microenvironment.

Example 9. SP-CTL cells were identified in vivo to kill primary gastric cancer cells

1) Cut the primary gastric cancer tissue sample into small pieces of tumor tissue with a diameter of 3 mm, and transplant it into the immunodeficient mouse NOD/SCID IL2rg-/- to construct a gastric cancer mouse model.

2) After 20 days, a SP-CTL virus-infected mixed cell with SP-CTL GFP positive cells of 4×10 ⁶ was injected into a mouse model of gastric cancer, and a second injection of 4×10 ⁶ SP-CTL virus infection was mixed for 25 days. The cells and the control group were tumor-bearing mice injected with GFP T cells, and three replicates were set for each experimental group;

3) On the 44th day after tumor transplantation, tumor tissue blocks were obtained from the mouse model of gastric cancer; the size comparison of the three replicated tumor tissue blocks in each experimental group is shown in Figure 14-A, for each experimental group. The mean weight results for the tumor tissue mass are shown in Figure 14-B.

From the above test results, it can be seen that the SP-CTL cells of the present invention can effectively eliminate primary gastric cancer cells in vivo in the case of primary tumor heterogeneity and immunosuppressive microenvironment.

Example 10, SP-CTL cells in vivo to identify primary osteosarcoma cells

1) Cut the original osteosarcoma tissue sample into small pieces of tumor tissue with a diameter of 3 mm, and transplant it into the immunodeficient mouse NOD/SCID IL2rg-/- to construct a mouse model of osteosarcoma.

2) 12 days after injection of osteosarcoma tumor mouse model SP-CTL GFP positive cells to SP-CTL 4 × 10 ⁶ virus infected cells were mixed, 14 second injection of 4 × 10 ⁶ SP-CTL infected The cells were mixed, and the control group was tumor-bearing mice injected with GFP T cells, and six replicates were set for each experimental group;

3) On the 38th day after tumor transplantation, tumor tissue blocks were obtained from the osteosarcoma mouse model; the size comparison images of six replicate tumor tissue blocks in each experimental group are shown in Figure 15-A, and each experimental group The average weight results for the tumor tissue mass are shown in Figure 15-B.

From the above test results, it can be seen that the SP-CTL cells of the present invention can effectively remove primary osteosarcoma cells in vivo in the case of primary tumor heterogeneity and immunosuppressive microenvironment.

Example 11, SC-CTL cells specifically recognize killer non-Hawking's lymphoma RL cells in vitro

1) SC-CTL GFP-positive cells and non-Hawkinson's lymphoma RL cells (cell lines are labeled with luciferase GL) at a cell number of 2:1, 1:1, 0.5:1, 0.25:1, 1:16 Proportional mixed co-culture (control group was added GFP T cells, each set of three duplicate wells);

2) After 18 hours, gently pipette a half volume of the medium supernatant, add an equal volume of luciferase substrate (concentration: 300 μg / ml), and mix;

3) The in vitro killing ratio of SC-CTL cells to RL-GL cells was detected by a multi-function microplate reader, and the results are shown in Fig. 16.

From the above test results, SC-CTL cells can specifically recognize and kill non-Hawkinson lymphoma cells in vitro.

Example 12, SC-CTL cells specifically recognize killer non-Hawking's lymphoma RL cells in vivo

1) 1×10 ⁵ RL-GL cells were subcutaneously transplanted into immunodeficient mouse NOD/SCID IL2rg-/- to construct a RL-GL tumor-bearing mouse model;

2) Two days later, RL-GL tumor-bearing mice were injected with SC-CTL virus-positive cells with a population of 1 × 10 ⁵ SC-CTL virus-infected mixed cells, and the control group was tumor-bearing mice injected with GFP T cells. Three experimental groups set up three repetitions;

3) On the 28th day after tumor transplantation, tumor tissue blocks were obtained from the mouse model of RL-GL tumor-bearing mice; the size comparison of two repeated tumor tissue blocks in each experimental group is shown in Figure 17-A. Tumor tissue of each experimental group The average weight result of the block is shown in Figure 17-B.

From the above test results, it is known that the SC-CTL cells of the present invention can effectively eliminate non-Hawsonson lymphoma cells in vivo.

Example 13, mSC-CTL cells specifically recognize killer primary osteosarcoma cells in vivo

Substitution of mouse extracellular domain + transmembrane region of CTLA4 (nucleotide sequence and amino acid sequence as shown in SEQ ID NO. 5, SEQ ID NO. 6) by molecular cloning means (h) extracellularization of SC-CTL Segment + transmembrane region, mSC-CTL was obtained. The purpose of this example was to evaluate the specific recognition and killing effect of mSC-CTL cells on primary osteosarcoma cells in vivo. The specific procedures are as follows:

1) Cut the original osteosarcoma tissue sample into small pieces of tumor tissue with a diameter of 3 mm and subcutaneously transplant it into the immunodeficient mouse NOD/SCID IL2rg-/- (2 small pieces per recipient mouse). Constructing a mouse model of osteosarcoma;

2) 20 days later, in the osteosarcoma mouse model, mSC-CTL GFP-positive cells were injected with mixed cells of m××10 ⁵ mSC-CTL virus, and the control group was tumor-bearing mice injected with GFP T cells. The group sets three repetitions;

3) Tumor tissue blocks were obtained from the osteosarcoma mouse model 44 days after tumor transplantation; the size of the tumor tissue blocks in each experimental group was shown in Fig. 18-A, and the tumor tissue blocks of each experimental group were The average weight results are shown in Figure 18-B.

From the above test results, it can be seen that the mSC-CTL cells of the present invention can effectively eliminate primary osteosarcoma cells in vivo in the case of primary tumor heterogeneity and immunosuppressive microenvironment.

Example 14, in combination with mSC-CTL and SP-CTL to identify killing tumor cells in vivo

Substitution of mouse extracellular domain + transmembrane region (nucleotide sequence and amino acid sequence as shown in SEQ. NO 5, 6) by mouse cloning means (h) extracellular domain + transmembrane region of SC-CTL , obtaining mSC-CTL; the purpose of this example is to evaluate the scavenging effect of SP-CTL and mSC-CTL on tumor cells, and evaluate SC-CTL recognizes the killing effect of mouse CD80+/CD86+ tumor helper cells (non-tumor cells) and evaluates the effect of this effect on tumor clearance. The specific procedures are as follows:

2) After 18 days, the lung cancer mouse model was divided into four groups, and SP-CTL virus-infected mixed cells (SP-CTL group) with SP-CTL GFP positive cells of 2×10 ⁵ were injected, and mSC-CTL GFP-positive cells were injected. 2 × 10 ⁵ SP-CTL virus-infected mixed cells (mSC-CTL group), and mixed 2×10 ⁵ SP-CTL GFP-positive cells and 2×10 ⁵ mSC-CTL GFP-positive cells (SP-CTL+) mSC-CTL combined application group), the control group was tumor-bearing mice injected with GFP T cells, and each experiment group was set up with three repetitions;

3) The volume of subcutaneous tumor mass of lung cancer was measured on the 18th, 21st, 24th, 27th, 30th, 33rd and 36th day after tumor transplantation. At different time points, the average volume of tumor tissue blocks in each experimental group is shown in Figure 19-A. Shown.

4) On the 38th day after tumor transplantation, tumor tissue blocks were obtained from the mouse model of lung cancer, and the average weight results of the tumor tissue blocks of each experimental group are shown in Fig. 19-B; the results show: SP- of the present invention CTL combined with mSC-CTL cells can effectively and synergistically eliminate primary lung cancer cells in vivo, and the effect is more significant than SP-CTL or mSC-CTL cells alone.

5) Take the tumor tissue blocks of SP-CTL, mSC-CTL, SP-CTL+mSC-CTL combined application group, grind and digest into single cells, and detect CD80, CD86, MDSC of mice in tumor tissue block by flow cytometry. The ratio of (CD11b+Ly6G+) cells, and the results are shown in Figures 20-A, 20-B, and 20-C.

From the above test results, the following conclusions can be drawn: mSC-CTL can not only identify killing tumor cells, but also recognize CD80+ or/and CD86+ cells (except tumor helper cells other than tumor cells), and mSC-CTL for CD80+/CD86+ The killing effect of tumor cells can effectively inhibit tumor growth. mSC-CTL combined with SP-CTL can simultaneously identify killer tumor cells and CD80+/CD86+ tumor-assisted cells, and block the effects of CD80+/CD86+ tumor-assisted cells on tumor cell growth, migration, drug resistance, immune escape, etc. Tumor elimination effect fruit.

Example 15. High expression of Lag3 ligand MHC II in leukemia and lymphoma cells (Fig. 21)

Flow cytometry was used to detect the expression of human B cell leukemia cell line Nalm6, B cell lymphoma cell line Raji and RL cell surface molecule human HLA-DR (HLA-DR is a key component of MHC II molecule). The lines all specifically express human HLA-DR gene, which specifically expresses MHC II molecules (Lag3 ligand), indicating that human B cell leukemia and B cell lymphoma tumor cells are potential target cells of SL-CTL.

Example 16. SL-CTL specifically recognizes tumor cells that secrete Lag3 ligand (Figures 22, 23)

1) SL-CTL cells were obtained by packaging (p. 1) lentivirus pWPXLd-SL-CTL-2A-EGFP plasmid and infecting human primary T cells.

2) Incubating the expanded SL-CTL cells with the target cells Nalm6-GL cells in a 96-well plate at a different E(Effector):T(Target) ratio of 2:1, 1:1, 1:2, 1: 4. Mix 1:8 and co-culture for 18 hours.

3) gently pipette half of the medium supernatant, add an equal volume of luciferase substrate (concentration: 300μg / ml), and mix;

4) The in vitro killing ratio of SL-CTL cells to A549-GL and K562-GL cells was detected by a multi-function microplate reader, and the results are shown in Fig. 22, respectively.

5) SL-CTL cells (blank control group GFP-T) and Nalm6-GL cells were co-cultured for 1:1 with 1:1, and the interleukin-2 (IL-2) and interference in the culture supernatant were detected by ELISA. The level of gamma (IFNγ) is shown in Figure 23. The results showed that SL-CTL cells were able to kill Nalm6 target cells more efficiently than wild T cells (GFP-T group), and SL-CTL cells secreted more inflammatory factors IL-2 and IFNγ.

Example 17, tumor cells in vivo can up-regulate the expression of its own TIM3 ligand Galectin9 (Figures 24, 25).

1) Detection of A549 cell line by flow cytometry does not express Galectin9;

2) A549 cells were transplanted into immunodeficient mice (such as NOD/SCID IL2rg-/-), 21 days later, The tumor block of the mouse was taken, ground and lysed into a single cell suspension, and the proportion of Galectin9-positive cells was detected, as shown in FIG. 24;

3) Three cells of human lung cancer samples from #1, #2, and #3 were transplanted into immunodeficient mice. After 60 days, the human lung cancer tumor tissue was taken from the mouse and ground into a single cell suspension. The proportion of Galectin9 positive cells in tumor tissue blocks was detected by flow cytometry as shown in FIG.

4) The data in this example show that tumor cells in vivo (closer to the tumor state in the patient) specifically up-regulate the expression of Galectin9 (Tim ligand) relative to the in vitro cultured tumor cell line, indicating in vivo status (including patients) Tumor cells of tumor cells in an in vivo state are potential specific target cells of ST-CTL cells.

Example 18, ST-CTL cells specifically recognize and kill tumor cells expressing TIM3 ligand in vivo (Fig. 26)

1) Cut the primary lung cancer tissue sample #2 into small pieces of tumor tissue with a diameter of 3 mm, and transplant it into the immunodeficient mouse NOD/SCID IL2rg-/- to construct a lung cancer mouse model.

2) 20 days after injection of lung cancer in a mouse model ST-CTL of GFP positive cells of 4 × 10 ⁶ ST-CTL infected cells were mixed, a second injection 25 days the number of GFP-positive cells in the SP- 4 × 10 ⁶ The CTL virus infects mixed cells, the control group is tumor-bearing mice injected with GFP T cells, and the blank (NC) group is tumor-free mice without treatment (no T cells are injected), and three replicates are set for each experimental group;

3) On the 45th day after tumor transplantation, tumor tissue blocks were obtained from three groups of experimental lung cancer mouse models; the tumor tissue mass was weighed, and the results are shown in Fig. 26.

4) It was found that ST-CTL cells can inhibit the growth of tumor cells in vivo.

The Applicant declares that the present invention describes the products, uses, and uses thereof of the present invention by the above embodiments, but the present invention is not limited to the above detailed uses and modes of use, that is, does not mean that the present invention must rely on the above detailed uses and uses. The way can be implemented. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

List of gene sequences involved in this article

SEQ ID NO.1 PD-1 signal peptide + extracellular domain + transmembrane region - CD28-TLR1/2-CD3ζ

SEQ ID NO.2 CTLA-4 signal peptide + extracellular domain + transmembrane region - CD28 intracellular segment - TLR1/2 intracellular segment - CD3

SEQ ID NO.3 TIM3 signal peptide + extracellular domain + transmembrane region - 4-1BB intracellular segment - TLR2 intracellular segment - CD3

SEQ ID NO.4 LAG3 signal peptide + extracellular domain + transmembrane region - 4-1BB intracellular segment - TLR2 intracellular segment - CD3

SEQ ID NO.5 mCTLA4 extracellular domain + transmembrane region

SEQ.ID NO.6 mCTLA4 extracellular domain + transmembrane region

Claims

A chimeric immunosuppressive checkpoint receptor molecule comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain and optionally a transmembrane domain are immunosuppressive assays A corresponding domain of a point receptor molecule or a genetic modification based on the domain, the intracellular domain being one or more immune activation signal domains.
The immunosuppressive checkpoint receptor molecule according to claim 1, wherein the immunosuppressive checkpoint receptor molecule is selected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, A2AR, B7H3, B7H4, BTLA, IDO, KIR, CD160, 2B4 (CD244) and VISTA (C10orf54);

Preferably, the immune checkpoint receptor molecule is PD-1, CTLA-4, LAG3 or TIM3.
The immunosuppressive checkpoint receptor molecule according to claim 1 or 2, wherein the intracellular domain comprises an immunostimulation signal intracellular domain combination and an optional signal peptide;

Preferably, the immune costimulatory signal combination comprises a TLR1 and/or TLR2 signal domain;

Further preferably, the intracellular domain is a combination of TLR1 and/or TLR2 and a signal domain such as 41BB and/or CD28;

Preferably, the optional signal peptide is a signal peptide corresponding to an immunosuppressive checkpoint receptor molecule.
The immunosuppressive checkpoint receptor molecule according to claim 3, wherein the intracellular domain further comprises a CD3 sputum intracellular domain;

Preferably, the TLR1 and/or TLR2 signal domain and optionally the 41BB and/or CD28 signal domain are arranged on the N-terminal side of the CD3 cell intracellular domain.
A nucleic acid encoding a chimeric immunosuppressive checkpoint receptor molecule of any of claims 1-4.
A nucleic acid construct comprising the nucleic acid of claim 5, and one or more control sequences operably linked thereto for directing expression of said chimeric immunosuppressive checkpoint receptor molecule in a host cell .
An expression vector comprising the nucleic acid construct of claim 6; preferably, the expression vector is a lentiviral expression vector.
A transformed cell comprising the nucleic acid of claim 5, or wherein the nucleic acid construct of claim 6 or the expression vector of claim 7 is transformed;

Preferably, the transformed cell is an immune cell, preferably an immune effector cell, further preferably a T cell, a B cell or an NK cell.
A method of producing the transformed cell of claim 8, comprising: introducing the nucleic acid of claim 5 into the genome of the cell, or transforming the nucleic acid construct of claim 6 or the method of claim 7 The step of the expression vector.
A chimeric immunosuppressive checkpoint receptor molecule according to any one of claims 1 to 4, a nucleic acid according to claim 5, a nucleic acid construct according to claim 6, or a method according to claim 7. The expression vector or the transformed cell of claim 8 is associated with the expression of a ligand corresponding to an immunosuppressive checkpoint receptor molecule, preferably PD-L1, B7.1, B7.2, Galectin-9, MHC II Use of the drug for the disease;

Preferably, the disease is a tumor expressing a ligand corresponding to an immunosuppressive checkpoint receptor molecule, preferably a tumor expressing PD-L1, B7.1, B7.2, Galectin-9, MHC II;

Further preferably, the tumor expressing the ligand corresponding to the immunosuppressive checkpoint receptor molecule, preferably the tumor expressing PD-L1, B7.1, B7.2, Galectin-9, MHC II is selected from the group consisting of lung cancer, leukemia, and mammary gland Cancer, prostate cancer, pancreatic cancer, liver cancer, melanoma, and non-melanoma skin cancer.